The proposed work is to continue studies of lipid metabolism in diverse bacteria some of which are human pathogens. Although many aspects of bacterial lipid metabolism were first worked out in the paradigm Escherichia coli, other bacteria differ from E. coli in various aspects. A focus will be on the last step of the fatty acid elongatin cycle, the enoyl-ACP reductase reaction, which is catalyzed by a set of very diverse proteins. Since fatty acids are an essential component of bacterial membrane lipids, several of these enzymes are the targets of antimicrobials. A second focus will be on the mechanisms of synthesis of unsaturated fatty acids, an essential component of the membrane lipids of most bacteria. A major puzzle is the mechanism of unsaturated fatty acid synthesis in the Clostridia, which include a number of serious human pathogens. Further investigations of the mechanisms of synthesis of the fatty acid-derived vitamins, biotin and lipoic acid, which are essential enzyme cofactors in all three domains of life, are proposed. Prior work provided the E. coli synthetic pathway in E. coli. However, some bacteria make these cofactors but lack one or more of the proteins used by. Current work is focused on the Firmicutes which include the Bacilli, Clostridia, Staphlococci and thus many important pathogens. In the case of lipoic acid synthesis Bacillus subtilis requires four proteins to synthesize this cofactor whereas E. coli required only two proteins. One of the essential proteins is a key subunit of an enzyme of single carbon metabolism and this interplay will be studied. The early part of biotin synthesis in Bacillus subtilis differs greatly from the pathway used by E. coli and this pathway will be dissected. A third pathway to be studied is that found in the a-proteobacteria (e.g., Brucella) which seems a variation of the E. coli pathway. Study of the mechanisms regulating the synthesis of the various biotin and lipoic acid synthetic pathways will also be done. The E. coli and Bacillus subtilis biotn regulatory systems are quite novel and not fully understood. Further study of these regulatory mechanisms is proposed. Finally, a new class of bacterial quorum sensing molecules the Diffusible Signal Factor (DSF) fatty acids were recently discovered. These are cis-2-enoic acids of medium chain length which trigger breakdown of the biofilms formed by diverse bacteria and some fungi. Prior work demonstrated how the Burkholderia cenocepacia DSF is synthesized, but that enzyme is not fully understood or conserved in some other DSF- producing bacteria. Study of these pathways and mechanisms will use a wide array of genetic, biochemical, molecular biological, chemical and structural approaches.